The long-range goal of this research is to define those biochemical events associated with hormonal regulation of gluconeogenesis. The immediate objectives focus on the control of hepatic pyruvate kinase in the rat and the relationship of this regulation with control of gluconeogenesis. Two specific aims are outlined. The first specific aim concerns the influence of adrenal glucocorticoids on regulation of pyruvate kinase by glucagon. Preliminary studies have shown that normal regulation of the enzyme by glucagon is lost in adrenalectomized rats. The regulation is restored toward normal by administration of dexamethasone to the isolated hepatocyte. Treating the hepatocyte with dexamethasone has also been shown to decrease cAMP-dependent phosphorylation of pyruvate kinase in cell-free extracts from those cells. The objective of this investigation will be to determine if glucocorticoids act by directly modifying pyruvate kinase or by regulating those enzyme involved in phosphorylation and dephosphorylation. All of these studies will be undertaken using isolated hepatocytes prepared from adrenalectomized rats. The second specific aim will investigate synthesis and turnover of hepatic pyruvate kinase. Presently, very little is known of those factors which act on the liver to control synthesis and degradation of the enzyme, although the levels of the enzyme in liver rapidly respond to dietary and hormonal changes in the animal. The proposed studies will specifically test the hypothesis that glucagon and glucocorticoids "trigger" proteolytic degradation of the enzyme. The roles of glucagon and insulin in modulating synthesis of the enzyme in rat liver will also be investigated. These studies will be carried out using intact rats as well as isolated hepatocytes maintained in culture. Synthesis and degradation of the enzyme will be studied using a high titer, monospecific antiserum against rat liver pyruvate kinase which was prepared in previous investigations into hormonal regulation of the enzyme.